PCR is one of the most widely used nucleic acid detection methods. It has various applications in the fields of research and molecular diagnosis of infectious diseases and genetic disorders. Among the many applications, blood-based PCR diagnoses are particularly ire demand.
On the one hand, the blood is a vital part of the circulatory system for the human body. It includes many types of host cells, including normal cells, diseased cells (e.g., circulating tumor cells), and, if the host is a pregnant mother, fetal cells, as well as genetic materials from such cells, such as microRNAs. Indeed, due to the minimum invasive nature, blood-based PCR is advantageous in some situations such as foetal diagnosis using a maternal blood sample without the risk of abortion associated with conventional amniotic fluid tests. Under certain circumstances, the blood also contains pathogens, such as viruses and bacteria. Accordingly, blood-based diagnoses can provide an immediate picture of what is happening in the human body at any given time. On the other hand, blood is the source for various pharmaceutical and blood products (e.g., whole blood, plasma, antibodies, and stem cells) that are used to improve the quality of life and to save lives in a variety of traumatic or pathological situations. These therapeutic uses of blood, plasma, and other blood-derived materials require that donations of these materials be as free as possible from contamination of diseased cells (e.g., tumor cells) or pathogens (e.g., HIV, HBV, and HCV). See, e.g., US Applications 20130316925, 20130157253, 20120329061, 20120070827, 20120034614, 20070281307, and 20070105121.
However, blood-based PCR diagnoses have been limited by a number of logistic and technical challenges. The challenges associated with nucleic acid diagnostics from biological samples are many folds. Complex biological samples, such as blood and cell lysates, have various components that can inhibit DNA polymerases used in PCR reactions. These components include hemoglobin, immunoglobulin G, lactoferrin, and proteases in blood. Although various procedures have been developed to purify samples before attempting PCR reactions, these steps are generally time-consuming, labor-intensive, and might not achieve the purification required for the subsequent PCR. In addition, nucleic acid-containing cells (e.g., white blood cells and fetal cells) account for only a small fraction of a blood sample, precious nucleic acid can be lost from the sample before the PCR reaction step.
When working with blood, one key point is that a blood sample should be collected in anticoagulants to prevent clotting since isolation of nucleic acids from clotted blood is not efficient and most of the cells will be lost in the clot. However, anticoagulants routinely used in blood sample collection, such as EDTA and heparin, interfere with or inhibit PCR. For example, heparin is highly negatively charged and will co-extract with DNA and thereby inhibit PCR reactions. See e.g., Garcia et al. J. Clin. Microbiol. April 2002 vol. 40 no. 4 1567-1568, and Yokota et al., Journal of Clinical Laboratory Analysis, Volume 13, Issue 3, pages 133-140, 1999.
Thus, there is a need for reagents, such as DNA polymerases, that are more resistant to the above-mentioned inhibitors and suitable for PCR reactions using blood samples.